Isoproturon is a widely used agricultural herbicide that can easily reach nearby water bodies through runoff, where it may affect non-target aquatic organisms. This study focuses on understanding the neurotoxic effects of sublethal Isoproturon exposure in the freshwater fish Cyprinus carpio (L.). Fish were exposed to two sublethal concentrations (1/5th and 1/10th of the 96-hour LC₅₀) for 7, 14, and 21 days. Neurotoxicity was evaluated by analyzing changes in acetylcholine (ACh) levels and acetylcholinesterase (AChE) activity in the brain. The results showed a clear and significant reduction in AChE activity, along with a corresponding increase in ACh levels, and these effects became more pronounced with higher concentrations and longer exposure periods. Such changes indicate disruption of normal nerve signal transmission, suggesting that Isoproturon interferes with neural functioning. The accumulation of ACh may be associated with altered ionic balance and stress within brain tissue. These findings demonstrate that even low, non-lethal levels of Isoproturon can adversely affect the nervous system of fish. Neurochemical changes observed in Cyprinus carpio highlight its usefulness as a sensitive bioindicator for detecting herbicide-induced neurotoxicity in freshwater ecosystems. The study emphasizes the need for careful regulation and monitoring of Isoproturon to prevent long-term neurological damage to aquatic organisms.